Multiple myeloma and chronic lymphocytic leukemia are composed of non- or slowly- replicating quiescent cell populations. Therefore, therapeutic approaches that do not target replicating DNA, but rather focus on transcription, translation, cellular bioenergy production, and critical molecular pathways may prove to be more effective. We have previously developed a halogenated ATP analog, 8-chloro-adenosine (8-CI-Ado) that has a unique RNA-directed mechanism of action. The special properties of this agent, two successful RAID awards, and availability of the GMP material has resulted in a clinical trial targeting patients with hematologic malignancies. The success of 8-CI-Ado stimulated our investigation of related analogs. We identified 8-amino-adenosine (8-NH2-Ado) and 8-azido-adenosine (8-N3-Ado) in this screen as having similar RNA-directed actions as the 8-CI-Ado halogenated congener. In preliminary studies, we have shown that 8-NH2-Ado actions are more potent and rapid than the halogenated congener. Most impressively, 8- NH2-Ado causes a massive accumulation of 8-NH2-ATP with a concomitant decrease in the endogenous ATP pools. In addition, there is a striking decrease in RNA synthesis, which is followed by a concurrent decrease in DNA synthesis. In this grant proposal, we will focus on the unique properties of 8-NH2-Ado and 8-N3-Ado in order to move these drugs forward to the clinical setting. In Aim I of this proposal, we will characterize the metabolism of these drugs, dissect their effects on mitochondria I function and the subsequent depletion of cellular bioenergy, and explore how these alterations may act on nucleic acid synthesis. In Aim II, we will further dissect the inhibitory actions toward RNA by examining changes in transcription and poly-adenylation. Finally, in Aim III, we will pursue the novel observation of decreased phosphorylation of key signaling pathways and how that impacts on apoptosis. Understanding the mechanisms underlying the actions of 8-modified adenosine analogs will allow for further rational drug design and lead to an identification of compounds that may complement the activity of these drugs in a therapeutic setting. Relevance to public health: These studies propose to investigate the mechanism of action of novel therapeutics that may be effective in treating slowly proliferating cancers of the blood such as multiple myeloma and chronic lymphocytic leukemia; cancers which are currently incurable.